Expression of polypeptides in yeast

ABSTRACT

DNA expression vectors capable, in a transformant strain of yeast, of expressing a polypeptide under the control of a genetically distinct yeast promoter, processes of forming transformant strains of yeast and transformed yeast strains are disclosed.

The contributions of certain of the coinventors hereof arose in thecourse of work funded in part by the United States Department of Healthand Human Services.

This application is a continuation application based on prior copendingapplication Ser. No. 08/383,668, filed Feb. 3, 1995 (abandoned), whichis a continuation of Ser. No. 08/198,535, filed on Feb. 18, 1994(abandoned), which is a continuation of Ser. No.08/089,419, filed Jul.9, 1993, (abandoned), which is a continuation of Ser. No.07/708,828,filed May 29, 1991 (abandoned), which is a continuation of Ser. No.07/349,918, filed May 9, 1989 (abandoned), which is a continuation ofSer. No. 07/284,774, filed Dec. 12, 1988 (abandoned), which is acontinuation of Ser. No. 07/173,008, filed Mar. 28, 1988 (abandoned),which is a continuation of Ser. No. 06/764,145, filed Aug. 9, 1985(abandoned), which is a continuation of Ser. No. 06/237,913, filed Feb.25, 1981 (abandoned).

FIELD OF THE INVENTION

This invention relates to the production, via recombinant DNAtechnology, of useful polypeptides in Saccharomyces cerevisiae (yeast),and to the means and methods of such production.

BACKGROUND OF THE INVENTION

The publications and other materials referred to herein to illuminatethe background of the invention and, in particular cases, to provideadditional detail respecting its practice are incorporated herein byreference, and, for convenience, are numerically referenced and groupedin the appended bibliography.

Recombinant DNA Technology

With the advent of recombinant DNA technology, the controlled microbialproduction of an enormous variety of useful polypeptides has becomepossible. Already in hand are bacteria modified by this technology topermit the production of such polypeptide products as somatostatin (1),the component A and B chains of human insulin (1), human proinsulin (2),thymosin alpha 1 (3), human growth hormone (4), human (5) and hybrid (6)leukocyte and fibroblast (7) interferons, as well as a number of otherproducts. The continued application of techniques already in hand isexpected in the future to permit bacterial production of a host of otheruseful polypeptide products, including other hormones, enzymes,immunogens useful in the preparation of vaccines, immune modulators andantibodies for diagnostic and drug-targeting applications.

The workhorse of recombinant DNA technology is the plasmid, anon-chromosomal loop of double-stranded DNA found in bacteria and othermicrobes, oftentimes in multiple copies per cell. Included in theinformation encoded in the plasmid DNA is that required to reproduce theplasmid in daughter cells (i.e., an "origin of replication") and,ordinarily one or more selection characteristics such as, in the case ofbacteria, resistance to antibiotics, which permit clones of the hostcell containing the plasmid of interest to be recognized andpreferentially grown under selective conditions. The utility of plasmidslies in the fact that they can be specifically cleaved by one or anotherrestriction endonuclease or "restriction enzyme", each of whichrecognizes a different site on the plasmid DNA. Thereafter heterologousgenes or gene fragments may be inserted into the plasmid by endwisejoining at the cleavage site or at reconstructed ends adjacent to thecleavage site. DNA recombination is performed outside the cell, but theresulting "recombinant" plasmid can be introduced into it by a processknown as transformation and large quantities of the heterologusgene-containing recombinant plasmid are then obtained by growing thetransformant. Moreover, where the gene is properly inserted withreference to portions of the plasmid which govern the transcription andtranslation of the encoded DNA information, the resulting expressionvehicle can be used to actually produce the polypeptide sequence forwhich the inserted gene codes, a process referred to as expression.

Expression is initiated in a region known as the promoter which isrecognized by and bound by RNA polymerase. The polymerase travels alongthe DNA, transcribing the information contained in the coding strandfrom its 5' to 3' end into messenger RNA which is in turn translatedinto a polypeptide having the amino acid sequence for which the DNAcodes. Each amino acid is encoded by a nucleotide triplet or "codon"within what may for present purposes be referred to as the "structuralgene", i.e., that part which encodes the amino acid sequence of theexpressed product. After binding to the promoter, the RNA polymerase,transcribes a 5' leader region of messenger RNA, then a translationinitiation or "start signal" (ordinarily ATG, which in the resultingmessenger RNA becomes AUG), then the nucleotide codons within thestructural gene itself. So-called stop codons are transcribed at the endof the structural gene whereafter the polymerase may form an additionalsequence of messenger RNA which, because of the presence of the stopsignal, will remain untranslated by the ribosomes. Ribosomes bind to thebinding site provided on the messenger RNA, and themselves produce theencoded polypeptide, beginning at the translation start signal andending at the previously mentioned stop signal. The resulting productmay be obtained by lysing the host cell and recovering the product byappropriate purification from other microbial protein or, in particularinstances, possibly by purification from the fermentation medium intowhich the product has been secreted.

Plasmids employed in genetic manipulations involved in the constructionof a vehicle suitable for the expression of a useful polypeptide productare referred to as DNA transfer vectors, Thus, employing restrictionenzymes and associated technology, gene fragments are ordered within theplasmid in in vitro manipulations, then amplified in vivo in thetransformant microbes into which the resulting, recombinant plasmid hasbeen `transferred`. A "DNA expression vector" comprises not only astructural gene intended for expression but also a promoter andassociated controls for effecting expression from the structural gene.Both transfer and expression vectors include origins of replication.Transfer vectors must and expression vectors may also include one ormore genes for phenotypic selection of transformant colonies.

Thus far, the useful products of expression from recombinant genes havefallen into two categories. In the first, a polypeptide having the aminoacid sequence of a desired end product is expressed directly, as in thecase of human growth hormone and the interferons referred to above. Inthe second, the product of expression is a fusion protein which includesnot only the amino acid sequence of the desired end product but also oneor more additional lengths of superfluous protein so arranged as topermit subsequent and specific cleavage away of the superfluous proteinand so as to yield the desired end product. Thus, cyanogen bromidecleavage at methionine residues has yielded somatostatin, thymosin alpha1 and the component A and B chains of human insulin from fusionproteins; enzymatic cleavage at defined residues has yielded betaendorphin (8).

A "biocompetent polypeptide", as that term is used herein, refers to aproduct exhibiting bioactivity akin to that of a polypeptide innatelyproduced within a living organism for a physiological purpose, as wellas to intermediates which can be processed into such polypeptides, as bycleavage away of superfluous protein, folding, combination (as in thecase of the A and B chains of human Insulin), etc.

Saccharomyces cerevisiae

The cells of Saccharomyces cerevisiae, or yeast, are, like those ofmammalian organisms, eukaryotic in nature as distinguished from theprokaryotic nature of bacteria. With regard to mechanisms for theexpression of genetic information, eukaryotes are distinguished frombacteria by:

(1) chromosomes which are organized in 140 base pair units, eachcontaining two molecules each of histones H2A, H2B, H3, and H4.

(2) Transcription of the protein-encoding gene by the alpha-amanitinsensitive RNA polymerase II.

(3) Post transcriptional addition of Gppp and polyadenylic acid to the5' and 3' termini of mRNA molecules.

(4) Transport of newly completed mRNA from the nuclei where they aretranscribed to the cytoplasm where they are translated.

(5) Some but not all eukaryotic genes contain intervening sequences(introns) which make them non-colinear with the corresponding matureMRNA molecule. The initial transcription products of these genes containthe intron sequence which is spliced out subsequently in the formationof a finished mRNA molecule.

The nucleotide sequences of all eukaryotic cells are transcribed,processed, and then translated in the context described above. There arereasons to believe that expression of eukaryotic genes may proceed withgreater efficiency in yeast than in E. coli because yeast is a eukaryotecell.

A number of workers have previously expressed, or attempted to express,foreign genes in yeast transformants. Thus, attempted expression from afragment comprising both a promoter and structural gene for rabbitglobin is reported (9) to have yielded partial mRNA transcripts,seemingly unaccompanied either by translation into protein or maturation(intron elimination) of the message. A gene coding for Drosophila GARtranformylas (yeast ADE8), an enzyme in the adenine synthesis pathway,is reported to have been expressed under the control of its own promoter(10). A number of yeast proteins have hitherto been expressed in yeastvia recombinant plasmids (see, eg., 12). In the experiments, as in theAde-8 case earlier discussed, expression occurred under the selectivepressure of genetic complementation. Thus, each expression product wasrequired for growth of the host strains employed, mutants whosechromosomal DNA was defective In the structural gene(s) from whichexpression occurred.

The availability of means for the production in yeast or proteins ofchoice could provide significant advantages relative to the use ofbacteria for the production of polypeptides encoded by recombinant DNA.Yeast has been employed in large scale fermentations for centuries, ascompared to the relatively recent advent of large scale E. colifermentation. Presently, yeast can be grown to higher densities thanbacteria, and is readily adaptable to continuous fermentationprocessing. Many critical functions of the organism, e.g., oxidativephosphorylation, are located within organelles, and hence not exposed tothe possible deleterious effects of the organism's overproduction offoreign proteins. As a eukaryotic organism, yeast may prove capable ofglycosylating expression products where important to enhancedbioactivity. Again, it is possible that as eukaryotic organisms, yeastcells will exhibit the same codon preferences as higher organisms,tending toward more efficient production of expression products frommammalian genes or from complementary DNA (cDNA) obtained by reversetranscription from, e.g., mammalian messenger RNA. Until the presentinvention, however, attempts to produce biocompetent expression productsother than those required for cellular growth have proven largelyunsuccessful.

BRIEF SUMMARY OF THE INVENTION

The present invention provides DNA expression vectors capable, intransformant strains of yeast, of expressing biologically competent(preferably pharmacologically active) polypeptides under the control ofgenetically distinct yeast promoters, the polypeptides being ordinarilyexogenous to yeast and other than those required for growth of thetransformant. The invention also provides DNA transfer vectors for thetransformation of yeast strains with genes encoding biocompetentpolypeptides, as well as novel yeast organisms and cultures thereofincorporating such vectors and methods for the formation of the same.The structural genes incorporated in the expression vectors andtransformant organisms of the invention are under the control ofgenetically distinct yeast promoters, i.e., promoters different fromthose evolutionarily associated with the subject structural genes.

The manner in which these and other objects and advantages of theinvention are obtained will become apparent from the detaileddescription which follows, and from the accompanying drawings in which:

FIG. 1 schematically illustrates the construction of a DNA transfervector having a single Eco RI restriction site for the subsequentinsertion of a yeast promoter and comprising both bacterial and yeastorigins of replication and selection phenotypes;

FIG. 2 schematically illustrates the construction of alcoholdehydrogenase promoter fragments for insertion into the transfer vectorof FIG. 1;

FIG. 3 illustrates the coding strand sequence and end points (904, 906,etc.) of a series of yeast promoter fragments made by digestion with theexonuclease Bal 31; and attachment of EcoRI molecular recombinationallinkers.

FIG. 4 schematically illustrates the insertion of yeast promoterfragments into the transfer vector of FIG. 1 and subsequent insertion(in two orientations) of a structural gene encoding human leukocyteinterferon D.

In the Figures and throughout, the letters A, T, C and G respectivelyconnote the nucleotides containing the bases adenine, thymine, cytosineand guanine. Only the coding strands of plasmids and gene fragments aredepicted. Though obviously not to scale, the representations of plasmidsdepict the relative position of restriction enzyme cleavage sites ("EcoRI", "HindIII" etc.) and other functions such as tetracycline resistance("Tc^(r) ") and ampicillin resistance ("Ap^(r) ").

DESCRIPTION OF PREFERRED EMBODIMENTS

Preferred embodiments of the invention are obtained by bringing anexogenous gene under the control of a yeast promoter carried by aplasmid suitable for the transformation of yeast. Essentially any yeaststrain suited for the selection of transformants may be employed. Inorder to achieve direct expression of the desired end product or anintermediate therefor, rather than a fusion comprising portions of theyeast protein whose expression is controlled by the promoter inwild-type strains, the parental plasmid is resected toward the promoterin the direction opposite that of transcription, so as to excise the ATGtriplet which initiates translation of mRNA encoding the yeast proteinreferred to.

An ordinarily exogenous gene, with its associated start signal, may thenbe inserted at the endpoint of the resection, and thus positioned fordirect expression under the control of the yeast promoter. This andother aspects of the invention are illustrated in the description ofpreferred embodiments which follow.

METHODS

Materials: All DNA restriction and metabolism enzymes were purchasedfrom New England Biolabs except for exonuclease Bal 31 and bacterialalkaline phosphatase, which were obtained from Bethesda ResearchLaboratories. DNA restriction enzyme and metabolic enzymes were used inconditions and buffers described by their respective manufacturers. ATPand the deoxynucleoside triphosphates DATP, dGTP, dCTP and DTTP werepurchased from PL Biochemicals. Eco RI, Bam HI, Hind III and Xho Ilinkers were obtained from Collaborative Research, Inc. α-³² P! wasobtained from New England Nuclear Corp.

DNA Preparation and Transformation: Purification of covalently closedcircular plasmid DNAs from E. coli (13) and yeast (14) plus thetransformation of E. coli (15) was as previously described.Transformation of yeast was as described by Hsiao and Carbon (16) withthe exception that 1.2M Sorbitol was used instead of 1.0M Sorbitol. E.Coli miniscreens were as described by (17).

Strains and Media: E. coli strain JA300 (thr leuB6 thi thyA trpC1117hsdM⁻ hsdR⁻ str^(R)) (18) was used to select for plasmids containingfunctional trpI gene. E. coli K-12 strain 294 (ATCC no. 31446) (19) wasused for all other bacterial transformation. Yeast strains RH218 havingthe genotype (a trpI gal2 suc2 mal CUPI) (20) and GM-3C-2 (α, leu 2-3,leu 2-112, trp 1-1, his 4-519, cyc 1-1, cyp 3-1) (21) were used foryeast transformations. Yeast strain RH 218 has been deposited withoutrestriction in the American Type Culture Collection, ATCC No. 44076.

M9 (minimal medium) with 0.25 percent casamino acids (CAA) and LB (richmedium) were as described by Miller (22) with the addition of 20 μg/mlampicillin (Sigma) after media is autoclaved and cooled. Yeast weregrown on the following media: YEPD contained 1 percent yeast extract, 2percent peptone and 2 percent glucose +3 percent Difco agar. YNB+CAAcontained 6.7 grams of yeast nitrogen base (without amino acids) (YNB)(Difco), 10 mg of adenine, 10 mg of uracil, 5 grams CAA, 20 gramsglucose and +30 grams agar per liter. The selection of ADH promoteractive fragments occurred on YEPGE plates containing 3 percent glyceroland 2 percent ethanol substituted for glucose in the YEPD formula.Leucine prototrophy was determined on plates containing 6.7 gms YNB, 20gms glucose, 50 mgs histidine and 50 mgs trytophan and 30 gms Difco agarper L.

Construction and Identification of ADH Promoter Deletions: pY9T6 wasdigested with Sau3A then run on a preparative 1 percent agarose gel. The1600 bp fragment containing the ADH promoter region was cut from thegel, electroeluted then purified on a diethylamino cellulose (DE52,Whatman) column before ethanol precipitation. Fragment DNA wasresuspended in DNA Polymerase I (Klenow fragment) buffer supplementedwith the four deoxyribonucleoside triphosphates in a final concentrationof 80 μM. Polymerase I was added and the thirty-minute room temperaturereaction was terminated by ethanol precipitation of the DNA. An equalmolar amount of BamHI and HindIII linker was added to the resuspendedSau3A fragment so that each linker was in a 20:1 molar excess to thelarge DNA fragment. T₄ DNA ligase was added and the 12 hour reactionoccured at 12 degrees centigrade. After ethanol precipitation andresuspension in the appropriate buffer, the DNA was digested with BamHI,then HindIII. The now larger promoter-containing fragment was purifiedaway from the unattached linkers by passage through a 10ml sizing columnbefore ethanol precipitation. This DNA fragment was then ligated inpBR322 previously isolated as missing the HindIII-to-BamHI restrictionfragment. E. coli strain RR1 was transformed to ampicillin resistanceusing part of this ligation mix. After quick screen analysis of a numberof recombinant plasmids, pJD221 which had the insert with the HindIIIlinker added to the end of the fragment closest to the ATG of the ADHstructural gene was isolated by plasmid preparation.

pJD221 was linearized with HindIII and the resulting fragment thansuccessively treated with exonuclease III and S₁ nuclease. The ends ofthese deleted plasmids were then made blunt using the Klenow fragment ofDNA Polymerase I (see procedure above). After ethanol precipitation theends of the DNA were ligated with XhoI linkers in a 12 hour reactionmixture. After digestion of resulting ligation mix with XhoI, plasmidsolution was run in a 0.5 percent preparative agarose gel. DNA bandswere cut from the gel, electroluted, then passed through a DE52 columnbefore ethanol precipitation. Linear plasmid was circularized using T4DNA Ligase. The resulting ligation mix was used to transform E. colistrain RR1 to ampicillin resistance. All such colonies were pooledtogether. The resulting single plasmid pool was cut with XhoI and BamHI,then run on a preparative 0.7 percent agarose gel. The 1500 bp bandscontaining the ADH promoter region were cut from the gel, electroelutedthen passed through a DE52 column before ethanol precipitation andligation into the vector pYecycI.increment.x+1. This plasmid hadpreviously been isolated from an agarose gel as having lost the XhoI toBamHI restriction fragment described in the Figure. The resultingligation was used to transform E. coli strain RRI to ampicillinresistance. Colonies were mixed for preparation of a plasmid pool whichwas then used to transform yeast strain GM-3C-2 to leucine prototrophy.Plasmids were then isolated from leucine prototrophs able to grow onglycerol plates. One plasmid, pACF 301, was found to contain a deletionextending toward the ATG of the ADH1 structural gene, leaving intact thefirst five triplets of the structural gene and the AC of the ACC of Thr₆(FIG. 2b). This plasmid was digested with XhoI then treated withexonuclease Bal3l for 15 and 30 seconds (two different aliquots).Resulting plasmids were pooled, ethanol precipitated and then treatedwith DNA Polymerase I (reaction described above) so that all DNA endswere made blunt. EcoRI linkers were then added to the DNA solution andligation allowed to proceed for 12 hours. After digestion with EcoRI andBamHI, ligation mix was run on a preparative agarose gel. A DNA bandabout 1500 bp in size was cut from the gel, electroeluted then passedthrough a sizing column before ethanol precipitation. This DNA was thenligated into the linear pBR322 DNA previously isolated as missing theEcoRI-to-BamHI restriction fragment. This ligation mix was used totransform E. coli strain 294 to ampicillin resistance. Plasmids isolatedfrom these colonies are referred to as the pGBn plasmid series.

Miniscreen analysis of a number of different recombinant plasmids fromthe pGBn plasmid series indicated that nine particular plasmids hadsmall Bal 31 generated deletions toward the ADH promoter region throughthe ATG of the ADH structural gene. All nine plasmids were digested withEcoRI, then end labeled by incubation with (α³² P)dATP and DNApolymerase I (conditions as described above). After ethanolprecipitation, seven plasmids were digested with AluI the nelectrophoresed on a 20 percent acrylamide--urea sequencing gel. ³²P--labelled plasmid DNAs from pGB904 and pGB906 were cut with BamHI thenrun on a preparative gel. Labelled fragments containing the ADH promoterregion were excised from the gel, electroluted, passed through a DE52column before ethanol precipitation. These two resuspended fragments(from plasmids pGB904 and pGB906) were then subjected to the G+A and T+Csequence specific degradation reactions described by Maxam and Gilbert(procedure 11 and 12 respectively (23)). These sequencing reactionproducts were electrophoresed along with labeled fragments from pGB905,pGB914, pGB917, pGB919 and pGB921 on the thin 20 percent acrylamidesequencing gel (described in the sequencing reference). Autoradiographywas as described. This procedure allowed the determination of the extentof deletion of ADH promoter region as this region had previously beensequenced using all four Maxam-Gilbert sequencing reactions (J.Bennetzen, Ph.D Thesis, University of Washington, 1980).

Expression Vector Construction: 10 μg of YRp7 (24-26) was digested withEcoRI. Resulting sticky DNA ends were made blunt using DNA Polymerase I(Klenow fragment). Vector and insert were run on 1 percent agarose(SeaKem) gel, cut from the gel, electroeluted and 2X extracted withequal volumes of chloroform and phenol before ethanol precipitation. Theresulting blunt end DNA molecules were then ligated together in a finalvolume of 50 μl for 12 hours at 12° C. This ligation mix was then usedto transform E. coli strain JA300 to ampicillin resistance andtryptophan prototrophy. Plasmids containing the TRPI gene in bothorientations were isolated. pFRW1 had the TRPI gene in the sameorientation as YRp7 while pFRW2 had the TRPI gene in the oppositeorientation.

10 μg of pFRW1 and 10 μg of YRp7 were digested with HindIII then run inseparate lanes on a 1 percent agarose gel. The large HindIII fragmentfrom the pFRW1 lane and the small fragment from the YRp7 lane wereeluted from the gel, extracted with phenol and chloroform, ethanolprecipitated, then ligated for 12 hours at 15° C. in a final volume of50 μl. This ligate mix was used to transform JA300 to tryptophanprototrophy and ampicillin resistance. Plasmid (pFRL4) containing asingle EcoRI site was then purified.

The pGBm plasmid series was digested with BamHI and EcoRI then run on a1 percent agarose gel. The ≅1500 bp promoter containing fragment fromeach lane was cut from the gel, electroeluted, then purified on a 10 mldiethylamino cellulose (Whatman) column before ethanol precipitation.

20 μg of pFRL4 was digested with BamHI and EcoRI then run on a 1 percentagarose gel. The large (≅5 kb) fragment was cut from the gel,electroeluted, 2X extracted with phenol and chloroform before ethanolprecipitation. 3 μg of this fragment was then separately ligated witheach of the promoter containing fragments for 12 hours at 15° C. in 50μl ligation mix. E. coli K-12 strain 294 was transformed with theligation mix to ampicillin resistance and plasmids from each of thesedifferent transformation mixtures were purified (pFRPn plasmid genes).

10 μg of pLeIF D (5) was digested with EcoRI then run on a 6 percentacrylamide gel. The 560 bp leukocyte interferon D gene was cut from thegel, electroeluted and 2X extracted with phenol/chloroform beforeethanol precipitation. This interferon gene was then ligated into theunique EcoRI site in the pFRPn plasmids previously cut with EcoRI andtreated with bacterial alkaline phosphatase. These vectors were thenused for BglII restriction analysis and yeast transformations.

Interferon Assay: Extracts of yeast were assayed for interferon bycomparison with interferon standards by the cytopathic effect (CPE)inhibition assay (27). Yeast extracts were prepared as follows: Five mlcultures were grown in YNB+CAA until reaching A₆₆₀ ≅1-2. Cells werecollected by centrifugation then resuspended in 600 μl of 1.2M sorbitol,10 mM KH₂ PO₄, pH=6.8 and 1 percent zymolyase 60,000 then incubated at30° C. for 30 min. Spheroplasts were pelleted at 3000 xg for 10 min.,then resuspended in 150 μl of 7M guanidine hydrochloride plus 1 mMphenylmethylsulfonylfouride (PMSF). Extracts were diluted 1,000 fold inPBS buffer (20 mM NaH₂ PO₄, pH=7.4, 150 mM NaCl, 0.5 percent BSA)immediately before the assay.

RESULTS Construction of a Vector for Insertion of a Series of PromoterFragments and for Insertion of a Gene to be Expressed

To design a plasmid vector for autonomous replication in yeast, it isnecessary to have both an origin of replication and a gene present forselection in yeast. Furthermore, the plasmid must contain a bacterialplasmid origin of replication and a means of selection in bacteria(e.g., an antibiotic resistance gene). With these requirements a plasmidcan be constructed and modified in vitro using recombinant DNAtechniques, amplified in bacteria, preferably E. coli, and finallytransformed into yeast.

Such a vector is shown in FIG. 1 and is designated YRp7 (24-26). Itcontains a chromosomal origin of replication from yeast (ars1) as wellas the TRP1 gene which codes for N-(5'-phosphoribosyl)-anthranilateisomerase (28). The TRP1 yeast gene can complement (allow for growth inthe absence of tryptophan) trp1 mutations in yeast (e.g., RH218, seeMethods) and can also complement the trpC1117 mutation of E. coli (e.g.JA300) (18). The plasmid is pBR322 (29) based so it also permits growthand selection in E. coli using antibiotic resistance selection.

Since it was necessary to clone into this vector BamHI/EcoRI restrictionfragments containing a yeast promoter, it proved convenient to firstremove one EcoRI site from the vector. This was done as shown in FIG. 1.The vector YRp7 was cut with EcoRI followed by filling in of the stickyEcoRI ends of both fragments with Klenow DNA polymerase I. The fragmentswere then blunt end ligated and the resulting DNA was used to transformE. coli JA300 to Trp+and ampicillin resistance (Ap^(R)). In such a wayplasmid pFRW1 was isolated with both EcoRI sites removed.

One EcoRI site was then restored to the plasmid in order that anEcoRI/BamHI fragment could be later cloned into the vector. This wasdone by cutting both YRp7 and pFRW1 with HindIII followed by theisolation of the fragments indicated. When the small HindIII fragment ofYRp7 was put together with the large HindIII fragment of pFRW1, pFRL4was obtained. It was selected for in E. coli JA300 using Trp+and ApRphenotypes.

Construction of Yeast Alcohol Dehydrogenase (ADH) Promoter Fragments

Since it is not known whether certain specific sequences in the leaderregion preceding structural genes are required for RNA polymerase IIbinding or what DNA is necessary for ribosome recognition (ribosomebinding sites) of the mRNA, promoter fragments from the ADH gene (ADCI)were obtained as described in FIG. 2.

The first step was to show that the 5'-leader DNA sequence of the ADHgene could be used to express another structural gene from yeast withoutits leader sequence (CYC1). Thus a plasmid which can complement a cyc1mutation in yeast can be used to isolate the ADH promoter fragment thatwill result in cyc1 expression. This promoter fragment could then beused to express other eukaryotic genes (eg., the Leukocyte Interferon Dgene).

As shown in FIG. 2, pY9T6 containing the ADC1 locus (Bennetzen, supra)was cut with Sau3A to isolate the 5'-flanking sequence of the ADH geneon an approximately 1600 bp fragment. The ATG translation start for theADH coding sequence is shown with the A at position +1, andtranscription goes from left to right as shown. This fragment was bluntended using Klenow DNA polymerase I followed by a ligation with amixture of BamHI and HindIII linkers. After cutting with BamHI andHindIII, the fragments were ligated with the large BamHI/HindIIIfragment of pBR322. The ligation products were used to transform E. colito Ap^(R) and the desired pJD221 was isolated from a transformant colonyusing a standard miniscreen procedure (see Methods). pJD221 was cut withHindIII and then with exonuclease III and S₁ nuclease to remove basepairs toward but not through the ATG of the ADH structural gene.

This procedure also removes base pairs in the opposite direction (towardthe EcoRI site) at approximately the same rate. The reaction wasdesigned so as to not remove the ATG of ADH since the ATG of CYC1 wasnot present in the fragment to be expressed under ADH promoter control.Therefore a complementation of cyc1 yeast would require a functionalADH1-CYC1 fusion protein.

The end-deleted products were treated with Klenow DNA polymerase I toensure blunt ends followed by the addition of XhoI linkers by blunt endligation. After XhoI cutting a circular plasmid was regenerated byligation. Plasmids containing gel-determined, properly sizedEcoRI-to-BamHI restriction fragments were digested with XhoI, thenligated with the large XhoI-to-BamHI restriction fragment of plasmidpYecyc 1 .increment.x+1. After amplification in E. coli RR1 resultingplasmids were used to transform a cyc1 cyc3 leu1 yeast strain to leucineprototrophy on minimal glucose plates. Growing colonies were patchedonto glycerol/ethanol plates. Yeast able to grow on such plates requirethe presence of functional cytochrome-c protein. This can only occur onthis plasmid if fragments containing ADH promoter deletions can initiate(in the correct reading frame) translation of the cytochrome c-codingregion. Plasmid pACF301 was isolated from one such transformant. Thejunction between ADH1 and CYC1 is shown at the bottom of FIG. 2b. Sixamino acid codons from the ADH sequence were present with 3 new aminoacid codons due to the XhoI linker, and the rest represented the CYC1structural gene. Thus the ADH promoter fragment is expressing a fusiongene product that produces a phenotypically active CYC1 gene fusionproduct.

In the construction of a yeast expression plasmid, it is desirable thatthe ATG codon of the non-yeast gene to be expressed be the one belongingto the same non-yeast gene rather than a vector ATG which would lead tothe synthesis of an undesired fusion protein. Therefore, it provedappropriate to remove nucleotides through the ATG of the ADH promoterfragment by another series of deletions and supply a new translationstart signal with the gene to be expressed. Since the functionality ofupstream DNA sequence (-1 to -1500) during the expression process is notknown, it was desirable to remove as little sequence as possibleupstream from the ATG and to try different fragments lacking both theinitially present ATG and various amounts of additional DNA sequence.

These additional promoter fragments were isolated as shown in FIG. 2b.pACF301 was cut with XhoI and Bal31. After blunt-ending, addition ofEcoRI linker, BamHI/EcoRI cutting, and sizing fragments; the correctsize class of fragments were ligated with EcoRI/BamHI-cut pBR322.Specific recloned ADH promoter fragments were isolated from plasmidsfrom various E. coli Ap^(R) transformants.

FIG. 3 shows the DNA sequences of the transcribed strand of 8 of theresulting, variously sized and numbered promoter fragments. The numberedlines show where the right end of the fragment ends and where the EcoRIlinker sequence begins.

The ends of fragments 904 and 906 were exactly determined by sequencing.The EcoRI sticky ends of these fragments were labelled with Klenow DNApolymerase using α-³² P-dATP. A sequencing gel was used to read from theA's into the linker through the Junction. The other 6 fragment ends wereapproximated to within about 1-2 base pairs by labelling as above,cutting with AluI, followed by sizing on the same denaturing gel.

Construction of Plasmids that Express Biologically Active LeukocyteInterferon D in Yeast

In order to optimize for successful expression of LeIF D in yeast, eightdifferent promoter fragments (FIG. 3) were ligated into the pFRL4 vectoras shown in FIG. 4.

The vector was designed to have ADH promoter transcription in the samedirection as TRP1 gene transcription (31). Since the LEIF D gene was tobe inserted in the EcoRI site and was not known to contain proper 3'termination and processing sequences for yeast recognition, the TRP1gene flanking sequence was aligned to perform these functions.

The resulting pFRPn series (where n is the promoter fragment number) wasobtained as shown. The preferred embodiment of these, pFRP6 in atransformant strain of E. coli 294, has been deposited in the AmericanType Culture Collection (ATCC no. 31814). These vectors were cut withEcoRI, alkaline phosphatase treated (to avoid prematurerecircularization), and ligated with the EcoRI LeIF D gene fragment. TheATG of this gene immediately follows the C of the EcoRI linker(GAATTCATG) as shown (FIG. 4).

Ampicillin resistant transformants of E. coli K-12 strain 294 werescreened to find plasmids containing both orientations of the LeIF Dfragment (pFRSn series--n refers to screening number). Orientations weredetermined by agarose gel electrophoresis using BglII digestion whichcuts both in the vector and in the LeIF D gene as shown.

Three of the plasmids demonstrated unpredicted restrictionpatterns.pFRS7 and pFRS35 have an extra BglII fragment at 560 bp. Thisresults from having two fragments of LeIF D in line with ADHtranscription. pFRS16 has no proper orientation fragment but has a 1700bp fragment which apparently resulted from the ligation of two vectorfragments together (two TRP1 containing "tails" together) with one LeIFD fragment in between two "heads" containing ADH promoter fragments.Thus in this ligation product the interferon gene is in the properorientation for expression by one of the ADH promoter fragments.

Evidence for Leukocyte Interferon D Expression in Yeast

First the E. coli 294 strains containing the various plasmids (pFRSn)were grown and extracts prepared (see Methods). No interferon activitywas observed in the extracts using the cytopathic effect inhibitionassay (see Methods). However, when plasmids were purified and used foryeast transformations by selection of TRP⁺ phenotype using yeast RH218(trp1 mutation), all plasmids with orientation I produced interferonactivity in yeast while no plasmids with orientation II producedInterferon.

Table 1 shows the results of interferon assays which measure antiviralactivity effects on VSV virus challenge of MDBK tissue culture cells(see Methods). Seven of the promoter fragments definitely express theLeIF D gene when the gene is in the proper orientation (I). This isdemonstrated by comparing units/(ml of extract) for the orientation Iplasmids with the orientation II plasmids. All orientation II plasmidsexpressed <1900 units/(ml of extract), a value 1 to 4 percent of thevalues for orientation I plasmids (actually background values areprobably much lower than this since the 1900 value is a function of theassay procedure).

                                      TABLE 1    __________________________________________________________________________    Interferon activity in yeast extracts          ADH                  Units.sup.b /l of                                      Cells.sup.c /l                                                 Units/cell          promoter Units.sup.a /ml                        Units/l of cells                               cells at Abs = 1                                      of culture                                           Pct..sup.c of cells                                                 containing                                                          Molecules.sup.d                                                          /cell    Orientation          fragment               Plasmid                   of extract                        ×10.sup.-6                               ×10.sup.-6                                      ×10.sup.-10                                           with plasmid                                                 ×10.sup.4                                                          containing    __________________________________________________________________________                                                          plasmid    I     904  pFRS3                    47,000                        1.4    1.2    2.1  19    3.5       53,000    I     905  pFRS7                    47,000                        1.4    0.54   3.4  30    1.4       21,000    I     913  pFRS16                   187,500                        5.6    2.0    5.1  32    3.4       51,000    I     906  pFRS12                   125,000                        3.8    1.8    2.7  19    7.4      110,000    I     906  pFRS36                   187,500                        5.6    2.3    3.0  16    12       180,000    I     915  pFRS23                   125,000                        2.1    0.81   3.6  21    2.8       42,000    I     921  pFRS35                   250,000                        7.5    2.8    4.5  18    9.2      140,000    I     919  pFRS34                    93,750                        2.8    1.4    2.2  17    7.6      110,000    II    904  pFRS2                   <1900                   22    II    905  pFRS6                   "                       23    II    913  pFRS17                   "                       36    II    906  pFRS11                   "                       13    II    915  pFRS22                   "                       46    II    921  pFRS26                   "                       25    II    919  pFRS33                   "                       31    __________________________________________________________________________     .sup.a Yeast cells were grown in 5 ml of YNB + CAA (Trp.sup.+  selection)     to an absorbance (Abs) of 1.2 to 2.8 at 660 mμ and spheroplasted with     zymolyase as described in Methods. The final pellet was resuspended in     0.15 ml of 7M guanidine HCl and 1 mM PMSF. The extracts were assayed usin     1/100 or 1/1000 dilutions in dilution buffer using an VSV virus challenge     of MDBK cells (see Methods).     .sup.b Units/liter of cells at Abs.sub.660 = 1 × 10.sup.-6 is a     normalization since different cultures were stopped between 1.2 to 2.8     Abs.sub.660.     .sup.c The number of yeast cells per culture was determined by dilution     and plating on YNB + CAA + tryptophan (50 μg/ml) plates. To find out     how many cells have plasmid (Trp.sup.+  complementation) cultures were     also plated on YNB + CAA (without tryptophan). Thus percentage of cells     containing plasmid was determined.     .sup.d Molecules/(cell containing plasmid) was calculated assuming     purified leukocyte interferon D has a specific activity of 2 ×     10.sup.8 units/mg (27) in the same assay and a molecular weight of 20,000     g/mole.

Since ars1 (chromosomal origin of replication)-containing plasmids havepreviously been shown to be unstable and lost in a high percentage ofthe cells even under selective maintenance pressure (24, 25), thepercent of cells containing the plasmid at the time of extractpreparation was measured. This was done by plating diluted cultures onplates with and without tryptophan. The results of this demonstrate thatthe plasmid is somewhat unstable in yeast (though not in bacteria), butcan be maintained by growth under selective pressure. These results alsoare evidence for the presence of the plasmid, since RH218 (trp1) yeastdo not grow on plates without tryptophan and since a revertant to TRP⁺would plate with equal efficiency on plates with and without tryptophan.Furthermore, the percentages of cells containing plasmid are similarcomparing yeast with orientation I and II plasmids. This suggests thatthe production of interferon in the yeast cell does not result inincreased instability of the plasmid due to interferon toxicity to thecell.

The fact that all the promoter fragments express interferon when up to32 bp are removed upstream from the ATG suggests that the DNA sequencein this region is relatively unimportant in transcription andtranslation. These results also suggest that precise spacing between thepromoter and the ATG may be relatively unimportant for expression inyeast.

In addition, Table 1 shows molecules/cell values which are very muchhigher than the 10,000 molecules/cell observed for interferon Dexpression in E. coli on a high copy plasmid with a strong promoter (trppromoter) (32). Assessment of this extreme difference (up to 18 fold) inmolecules per cell should recognize that the yeast cell volume isprobably 2 orders of magnitude higher than that of E. coli; however, theamount of expression from only 1-2 copies of the yeast plasmid versusthe high copy number of plasmids producing interferon in E. coli isdramatic.

Comparison of the Size of Interferon Produced in Yeast versus E. coli

Since the interferon gene uses its own ATG-initiation codon and sincethe alcohol dehydrogenase ATG has been removed in the construction, onewould expect to find that the interferon expressed in yeast is the samesize as the interferon in E. coli (32). SDS-polyacrylamide gelelectrophoresis was accordingly done on an E. coli extract containinginterferon D versus a yeast extract containing interferon D. Afterrunning the gel, two lanes containing yeast extract versus E. coliextract were simultaneously sliced. The slices were put into assaydilution buffer and left at 4° C. for 3 days. Interferon assays werethen performed to compare sizes of the peptides. Both appear to be about20,000 daltons, the size expected for interferon D. However, there doesappear to be a slight difference in the molecular weights, with yeastinterferon D being about 7 percent larger, possibly owing toglycosylation. Despite the size difference, the products of yeastexpression exhibited interferon activity (Table 1).

The preceding data clearly demonstrates that a yeast 5'-flanking DNAsequence, without the translation start signal of the structural gene,can efficiently promote the expression of an inserted mammalian or otherstructural gene for a biocompetent polypeptide, and do so without theaid of selective pressure for the product of expression (i.e., theexpression product is not required for cell growth).

The availability of yeast promoter-containing plasmids (pFRPn series)having both yeast and bacterial phenotypical genes and origins ofreplication, and a site downstream from the promoter convenient for theinsertion of translation start- and stop-bearing structural genespermits the creation of DNA expression vectors for a wide variety ofpolypeptides. Thus, into such a site may be inserted, for example,structural genes for both normal (5) and hybrid (6) human leukocyteinterferons, fibroblast interferon (7), somatostatin or the A or Bchains of human insulin (1), human proinsulin (2), thymosin alpha 1 (3),human growth hormone (4) and, indeed, virtually any other biocompetentpolypeptide.

Following expression, product may be extracted and purified as in thecase of bacterial expression, mutatis mutandis.

It will be appreciated that the invention Is not limited in itsapplication to the particular expression vector exemplified above. Forexample, use of the so-called two micron origin of replication wouldprovide additional stability, making unnecessary resort to selectivepressure for maintenance of the plasmid in the yeast cell, particularlyif the host strain is CIR+!, i.e., contains normal two micron plasmid(33). Such an expression vector would be stable in yeast in the richmedium ordinarily best for large scale fermentations. At the same time,use of the two micron origin of replication could significantly increaseplasmid copy number in each cell.

Stability of the expression vector in yeast may also be enhanced byinclusion within the plasmid of a yeast centromere (34), an elementinvolved in maintenance of the yeast chromosome. The resulting plasmidwill behave as a minichromosome, such that selective pressure will notbe required during growth or maintenance of the plasmid. As many as 17different yeast centromeres have been identified to the present date.

Transcription terminators other than that present on the TRP1 gene maybe employed, e.g., other 3'-flanking sequences from yeast such as the3'-flanking sequence contained on a Hinc II-BamH1 fragment of the ADH 1gene.

Optimization may also result from alteration of the sequence between theyeast promoter fragment and the inserted gene fragment. Thus, an A(adenine base) is found at position -3 (the third base before thetranslation start signal) of all twenty different mRNA-coding yeastgenes heretofore sequenced. A variety of means (eg., use of linkers) forincluding such an element in the plasmids of the invention will appearto those skilled in the art.

Of course, promoters other than the ADH promoter exemplified above maybe employed in variants of the invention. For example, the promoter ofthe yeast 3-phosphoglycerate kinase gene may be employed, doubtlessincreasing expression levels significantly over those observed for theADH system. Again, one or more of the promoters for yeastglyceraldehyde-3-phosphate dehydrogenase may be employed. This system isnonfunctional in the absence of glucose, but induced 200-fold in itspresence, and could accordingly be employed for fine control ofexpression.

From the foregoing, it will be apparent that the invention provides newmeans for the expression of valuable polypeptides. In particularInstances, efficiency of expression relative to that in recombinantbacteria may result from the different codon usage patterns as betweenyeast and bacteria, such that eukaryotic genes may be better expressedin yeast. The yeast expression systems of the invention may also provideadvantage in the glycosylation of biocompetent polypeptides, an abilitybacteria lack. The glycosylation system of yeast is very similar to thatof higher eukaryotes, and glycosylation may prove to have profoundeffects on the functions of proteins.

As will be apparent to those skilled in the art in the light of theforegoing discussion, the invention is not to be limited to thepreferred embodiments thereof exemplified above, but rather only to thelawful scope of the appended claims.

BIBLIOGRAPHY

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We claim:
 1. A process of forming a transformant of a yeast straincapable of expressing a polypeptide ordinarily exogenous to yeast,comprising transforming said yeast strain with a DNA transfer vectorthat comprises:(a) bacterial and yeast origins of replication and genesfor phenotypic selection of both bacterial and yeast moietiestransformed with said vector; (b) a DNA fragment comprising a structuralgene encoding said polypeptide; (c) a yeast promoter geneticallydistinct from said structural gene obtained from the about 1500 bp DNAsequence 5' flanking the start signal of a yeast structural gene; and(d) translation start and stop signals for said structural gene encodingsaid polypeptide that are positioned so that said structural geneencoding said polypeptide is under the control of said promoter whilemaintaining said origins of replications and genes for phenotypicselection.
 2. The process of claim 1, wherein the vector furthercomprises a transcription terminator located between the 3' end of acoding strand of said structural gene encoding said polypeptide and saidorigins of replication.
 3. The process of claim 1, wherein the DNAfragment comprising said structural gene further comprises a translationstart signal and a translation stop signal.
 4. The process of claim 1,wherein the gene for phenotypic selection of bacteria encodes apolypeptide that confers antibiotic-resistance and wherein the gene forphenotypic selection of yeast encodes a polypeptide that complements amutation carried by the yeast strain to be transformed.
 5. The processof claim 4, wherein the yeast strain is Saccharomyces cerevisiae.
 6. Theprocess of claim 5, wherein the yeast strain is strain RH
 218. 7. Theprocess of claim 1, wherein the amino acid sequence of said polypeptideis selected to correspond to the amino acid sequence of a polypeptideselected from the group consisting of the normal and hybrid humaninterferons, human proinsulin, the A and B chains of human insulin,human growth hormone, somatostatin, and thymosin alpha
 1. 8. The processof claim 1, wherein the polypeptide is the A or B chain of humaninsulin.
 9. The process of claim 1, wherein the yeast promoter includesa transcription start site of the yeast structural gene.
 10. A processof producing a polypeptide comprising culturing a yeast straintransformed by the process of claim 1, and recovering said polypeptide.11. The process of claim 10, wherein the amino acid sequence of saidpolypeptide is an intermediate from which the A and B chains of-humaninsulin can be obtained by cleavage away of superfluous amino acids. 12.A DNA expression vector, capable in a transformant strain of yeast ofexpressing EL polypeptide ordinarily exogenous to yeast, said vectorcomprising bacterial and yeast origins of replication and a yeastpromoter from the about 1500 bp DNA sequence 5' flanking the startsignal of a yeast structural gene, said yeast promoter being capable ofdirecting the expression of a gene encoding said polypeptide.
 13. TheDNA expression vector of claim 12, which further comprises a geneencoding a polypeptide under the control of the yeast promoter.
 14. TheDNA expression vector of claim 13, wherein the promoter is resected freeof the yeast structural gene and the start signal of the yeaststructural gene, and the gene encoding the polypeptide is located at theendpoint of the resection.
 15. The DNA expression vector of claim 13,which further comprises a transcription terminator following the geneencoding the polypeptide in the 3' direction in which the gene istranscribed.
 16. The DNA expression vector of claim 15, wherein thepolypeptide is a mammalian polypeptide.
 17. The DNA expression vector ofclaim 13, which further comprises a gene encoding a polypeptide forphenotypic selection of a yeast transformant.
 18. The DNA expressionvector of claim 17, which further comprises a gene for encoding apolypeptide for phenotypic selection in bacteria.
 19. The DNA expressionvector of claim 18, wherein the gene encoding the polypeptide forphenotypic selection in bacteria comprises a gene encoding a polypeptidethat confers antibiotic resistance and the gene encoding the polypeptidefor phenotypic selection of the yeast transformant comprises a geneencoding a polypeptide that complements a mutation carried by the yeaststrain to be transformed with the expression vector.
 20. The DNAexpression vector of claim 13, wherein the polypeptide comprises theamino acid sequence of a polypeptide having the activity of apolypeptide selected from the group consisting of normal and hybridhuman interferons, human proinsulin, the A and B chains of humaninsulin, human growth hormone, somatostatin, and thymosin alpha
 1. 21.The DNA expression vector of claim 13, wherein the polypeptide is the Aor B chain of human insulin.
 22. The DNA expression vector of claim 13,wherein the polypeptide is a mammalian polypeptide.
 23. The DNAexpression vector of claim 13, wherein the yeast promoter comprises apromoter for the yeast 3-phosphoglycerate kinase,glyceraldehyde-3-phosphate dehydrogenase, or alcohol dehydrogenasestructural genes.
 24. The DNA expression vector of claim 13, wherein thepromoter is a yeast alcohol dehydrogenase promoter.
 25. The DNAexpression vector of claim 13, wherein the origin of replication is the2 micron yeast plasmid origin of replication.
 26. The DNA expressionvector of claim 13, wherein the structural gene is a yeast chromosomalgene.
 27. The DNA expression vector of claim 13, wherein the yeastpromoter further comprises a yeast transcription start site.
 28. Aprocess of producing a polypeptide comprising transforming a yeaststrain with the DNA expression vector of claim 12, culturing thetransformed yeast strain, and recovering said polypeptide.
 29. A yeaststrain comprising a gene encoding a polypeptide ordinarily exogenous toyeast under the control of a yeast promoter from the about 1500 bp DNAsequence 5' flanking the start signal of a yeast structural gene. 30.The yeast strain of claim 29, wherein the yeast promoter is resectedfree of the yeast structural gene and the gene encoding the polypeptideis located at the endpoint of the resection.
 31. The yeast strain ofclaim 29, wherein the polypeptide has the amino acid sequence of apolypeptide having the activity of a polypeptide selected from the groupconsisting of normal and hybrid human interferons, human proinsulin, theA and B chains of human insulin, human growth hormone, somatostatin, andthymosin alpha
 1. 32. The yeast strain of claim 29, wherein thepolypeptide is the A or B chain of human insulin.
 33. The yeast strainof claim 29, wherein the polypeptide is a mammalian polypeptide.
 34. Ayeast strain capable of expressing a polypeptide ordinarily exogenous toyeast produced by a process comprising transforming said yeast strainwith a DNA transfer vector comprising:(a) bacterial and yeast origins ofreplication and genes encoding polypeptides for phenotypic selection ofboth bacterial and yeast moieties transformed with said vector; (b) aDNA fragment comprising a structural gene encoding said polypeptide; (c)a DNA fragment comprising a yeast promoter from the about 1500 bp DNAsequence 5' flanking the start signal of a yeast structural gene; (d)translation start and stop signals for said structural gene encodingsaid polypeptide positioned such that said structural gene for saidpolypeptide is under the control of said yeast promoter, whilemaintaining said origins of replication and genes encoding polypeptidesfar phenotypic selection.
 35. The yeast strain of claim 34, wherein saidyeast promoter is resected free of said yeast structural gene and thegene encoding said polypeptide is located at the endpoint of theresection.
 36. The yeast strain of claim 34, wherein said polypeptidecomprises the amino acid sequence of a polypeptide having the activityof a polypeptide selected from the group consisting of normal and hybridhuman interferons, human proinsulin, the A and B chains of humaninsulin, human growth hormone, somatostatin, and thymosin alpha
 1. 37.The yeast strain of claim 34, wherein the yeast promoter furthercomprises a yeast transcription start site.
 38. A process of producing apolypeptide comprising culturing the yeast strain of claim 34, andrecovering said polypeptide.